Temporal Analysis of the Nuclear-to-cytoplasmic Translocation of a Herpes Simplex Virus 1 Protein by Immunofluorescent Confocal Microscopy

Summary

ICP0 undergoes nuclear-to-cytoplasmic translocation during HSV-1 infection. The molecular mechanism of this event is not known. Here we describe the use of confocal microscope as a tool to quantify ICP0 movement in HSV-1 infection, which lays the groundwork for quantitatively analyzing ICP0 translocation in future mechanistic studies.

Abstract

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is responsible for the proteasomal degradation of host restrictive factors and the subsequent viral gene activation. ICP0 contains a canonical nuclear localization sequence (NLS). It enters the nucleus immediately after de novo synthesis and executes its anti-host defense functions mainly in the nucleus. However, later in infection, ICP0 is found solely in the cytoplasm, suggesting the occurrence of a nuclear-to-cytoplasmic translocation during HSV-1 infection. Presumably ICP0 translocation enables ICP0 to modulate its functions according to its subcellular locations at different infection phases. In order to delineate the biological function and regulatory mechanism of ICP0 nuclear-to-cytoplasmic translocation, we modified an immunofluorescent microscopy method to monitor ICP0 trafficking during HSV-1 infection. This protocol involves immunofluorescent staining, confocal microscope imaging, and nuclear vs. cytoplasmic distribution analysis. The goal of this protocol is to adapt the steady state confocal images taken in a time course into a quantitative documentation of ICP0 movement throughout the lytic infection. We propose that this method can be generalized to quantitatively analyze nuclear vs. cytoplasmic localization of other viral or cellular proteins without involving live imaging technology.

Introduction

Herpes simplex virus 1 (HSV-1) causes a wide range of mild to severe herpetic diseases including herpes labialis, genital herpes, stromal keratitis, and encephalitis. Once infected, the virus establishes a lifelong latent infection in ganglia neurons. Occasionally, the virus can be reactivated by various reasons such as fever, stress, and immune suppression¹, leading to recurrent herpes infection. Infected cell protein 0 (ICP0) is a key viral regulator crucial for both lytic and latent HSV-1 infection. It transactivates downstream virus genes via counteracting the host intrinsic/innate antiviral defenses^2,3. ICP0 has an E3 ubiquitin ligase activity, which targets several cell factors for proteasome-dependent degradation³. It also interacts with various cell pathways to regulate their activities and subsequently to offset host antiviral restrictions³. ICP0 is known to locate at different subcellular compartments as the infection proceeds^3,4,5. The protein has a lysine/arginine-rich nuclear localization signal (NLS) located at residues 500 to 506⁶. Upon de novo synthesis at early HSV-1 infection, ICP0 is immediately imported into the nucleus. It is first detected at a dynamic nuclear structure termed nuclear domain 10 (ND10)⁷. The E3 ubiquitin ligase activity of ICP0 triggers the degradation of ND10 organizer proteins, promyelocytic leukemia (PML) protein, and speckled protein 100 kDa (Sp100)^8,9,10. After the loss of organizer proteins, ND10 nuclear bodies are dispersed and ICP0 is diffused to fill the entire nucleus^4,11.

Interestingly, after the onset of viral DNA replication, ICP0 disappears from the nucleus. It is solely found in the cytoplasm, suggesting the occurrence of a nuclear-to-cytoplasmic translocation late in HSV-1 infection^4,12. The requirement of the DNA replication implies the potential involvement of a late viral protein(s) in facilitating the cytoplasmic translocation of HSV-1 ICP0^4,12. Apparently ICP0 trafficking among different compartments during infection empowers ICP0 to modulate its interactions to various cellular pathways in a spatial-temporal fashion, and therefore coordinate its multiple functions to fine tune the balance between the lytic and latent HSV-1 infection¹³. To better understand ICP0 multifunctionality and the coordination of ICP0 functional domains throughout the lytic infection, we carefully dissected the molecular basis of the dynamic ICP0 translocation¹². To conduct the mechanistic studies previously reported¹², we have applied an immunofluorescent staining method to visualize ICP0 subcellular localization at different infection status under confocal microscope. We have also developed a quantitative protocol to analyze the nuclear vs. cytoplasmic distribution of ICP0 using the confocal software. The population of HSV-1 infected cells was tabulated throughout the infection phases and the trends of ICP0 movement were analyzed, under different biochemical treatments¹². Here we describe the detailed protocol that documents ICP0 translocation in HSV-1 infection. We propose that this method can be adopted as a general method to study the nuclear vs. cytoplasmic translocation for other viral or cellular proteins, which can serve as an alternative to live imaging when the live imaging technique is inapplicable due to problems such as labeling method, signal intensity, or protein abundance.

Protocol

1. Cell Seeding and Virus Infection

1. At 20–24 h before the virus infection, seed 5 x 10⁴ of human embryonic lung (HEL) fibroblast cells or other cells to be examined on a 4-well 11 mm staggered slide in growth medium (Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS)). Incubate the cells at 37 °C with 5% carbon dioxide (CO₂).
   NOTE: Each well should have 70-80% cell confluency at the time of infection.
2. On the next day, remove the growth medium and infect the cells with viruses in Medium-199 at a range of 4–10 pfu/cell. Incubate virus-infected cells for 1 h at 37 °C. Keep shaking the slide during the incubation period.
3. After the 1 h incubation, remove Medium-199 and supplement with growth medium.
   NOTE: Drugs that interfere with different infection phases can be added at this step or prior to viral absorption.
4. Incubate the virus-infected cells at 37 °C with 5% CO₂ for various lengths of infection period.

2. Fixation and Permeabilization

1. At proper infection time, quickly wash the infected cells with phosphate-buffered saline (PBS) 3 times and add 200 μL of 4% paraformaldehyde freshly prepared in PBS. Incubate the cells with paraformaldehyde for 8–10 min at room temperature to fix the cells in each well.
2. Aspirate paraformaldehyde and wash the wells with 200 μL of PBS for 3 times. Completely aspirate PBS after the 3^rd wash.
3. Add 100 μL of 0.2% non-ionic surfactant to each well to permeabilize the cells for 5–10 min.
4. Aspirate the non-ionic surfactant and wash the wells with 200 μL of PBS for 3 times.

3. Immunofluorescent Staining

1. Completely aspirate PBS and add 200 μL of blocking buffer (1% bovine serum albumin (BSA) and 5% horse serum in PBS) in each well and incubate at room temperature for 1 h or at 4 °C overnight.
2. Add experimentally determined concentration of primary antibody (rabbit anti-ICP0 polyclonal antibody¹²) in blocking buffer and incubate primary antibody at room temperature for 2 h or at 4 °C overnight.
3. Wash with blocking buffer 3 times with 10 min incubation. Add Alexa 594-conjugated goat anti-rabbit secondary antibody (1:400 diluted in blocking buffer) and incubate the slides at room temperature for 1 h. Then wash the slides 3 times with blocking buffer at 10 min interval.
4. Finally wash the slide once with PBS to remove residual BSA and horse serum.
5. Add one drop of antifade mounting medium with 4',6-diamidino-2-phenylindole (DAPI) to mount the slide and seal it with coverslip using transparent nail polish.

4. Confocal Imaging

1. With a confocal microscope, set the wavelength at 590–650 nm for Alexa 594 and 410–520 nm for DAPI. Select image format at 1024 x 1024 and line average of 8 to acquire high resolution images.
2. Analyze each well on the 4-well slide under confocal microscope. Acquire representative cell images under the 100X objective, as shown in Figure 1 and Figure 2.
3. For counting large number of cells, take images of consecutive fields under the 40X objective.
   NOTE: It requires 5–10 images to accumulate over 200 infected cells from each time point of each infection.
4. In each experiment, take pictures with constant confocal parameters for all samples need to be compared.

5. Analyzing Nuclear vs. Cytoplasmic Distribution

1. Open project with the confocal application software. Select an image from which cells need to be tabulated for nuclear vs. cytoplasmic distribution of ICP0.
2. Click the tab "Quantity" from top menu and select "sort ROIs" from tools menu.
3. Draw a longitudinal line across the cell to be analyzed by selecting "Draw line" from top menu.
   NOTE: Histogram will appear showing the fluorescence intensity along the line for both ICP0 and DAPI. In the histogram, blue line represents DAPI pixels and marks the boundary of the nucleus whereas the red line represents ICP0 pixels.
4. Based on background staining, set up a constant threshold for ICP0 intensity to analyze ICP0 subcellular distribution in each experiment.
   1. As exemplified in Figure 2, if the red signal on average is below the threshold in the nuclear region but is above the threshold beyond the blue boundary, categorize the red signal as predominantly located in the cytoplasm.
   2. If the red signal is above the threshold throughout the nucleus and beyond the boundary of blue signal, group the red signal as nucleus plus cytoplasmic localization.
   3. If the red signal is above the threshold in the nucleus but on average is below it outside the boundary of blue signal, group the red signal as nuclear localization.
5. Tabulate more than 200 infected cells from each sample at different infection time and plot in bar graph to illustrate ICP0 movement according to time (Figure 3).

Results

To understand the molecular basis and biological functions of ICP0 trafficking during HSV-1 infection, we use an immunofluorescent microscopy method to analyze ICP0 subcellular distribution at different infection phases. Figure 1 shows the representative cells with distinctive ICP0 localization as the infection progresses. To quantify the nuclear-to-cytoplasmic translocation of ICP0, we analyze ICP0 distribution relative to the nucleus by categorizing infecte...

Discussion

This protocol has been used to study the nuclear-to-cytoplasmic translocation of HSV-1 ICP0. ICP0 undergoes subcellular trafficking during HSV-1 infection (Figure 1). Likely, ICP0 interacts with various cell pathways to carry out different functions at different locations. This enables ICP0 to fine tune its multiple functions in the tug-of-war with human host¹³. However, how ICP0 coordinates the multiple functions in a spatial-temporal manner has not been well studied...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cells and viruses
Human Embryonic Lung fibroblasts (HEL Cells)	Dr. Thomas E. Shenk (Princeton University)		HEL cells were grown in DMEM supplemented with 10% FBS
HSV-1 viral Stock (Strain F)	Dr. Bernard Roizman Lab
Medium
Dulbecco’s modified Eagle’s medium (DMEM)	Invitrogen	11965-092
Fetal Bovine Serum (FBS)	Sigma	F0926-500ml
Medium-199 (10x)	Gibco	11825-015
Reagents
4- well 11 mm staggered slide	Cel-Line/Thermofisher Scientific	30-149H-BLACK
16% Paraformaldehyde solution(w/v) Methanol free	Thermo Scientific	28908
Triton X-100	Fisher reagents	BP151-1C0
Bovine Serum Abumin (BSA)	Calbiochem	CAS 9048-46-8
Horse Serum	Sigma	H1270
Phosphate Buffered Saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, pH7.4)	Dr. Haidong Gu lab
NaCl	Fisher Bioreagent	BP358-212
KH2PO4	Fisher Bioreagent	BP362-500
KCl	Fisher Scientific	BP366-500
Na2HPO4	Fisher Bioreagent	BP332-500
Blocking buffer (PBS with 1% BSA and 5% Horse serum )	Dr. Haidong Gu lab
Rabiit Anti-ICP0 antibody	Dr. Haidong Gu lab
PML (PG-M3)-Mouse monoclonal IgG	santa Cruz Biotechnology	SC-966
Alexa Fluor 594-goat anti-rabbit IgG	invitrogen	A11012
Alexa Fluor 488-goat anti-mouse IgG	invitrogen	A11001
Vectashield Mouting medium with DAPI	Vector laboratories	H-1200
Pasteur pipette	Fisher Brand	13-678-20D
Nail Polish	Sally Hansen
Equipment
Confocal Microscope	Leica SP8
Confocal Software	Leica LAS X Application suite
Excel software	Microsoft Excel
HERAcell 150i CO2 incubator	Thermo Scientific	Order code 51026282